Project Summary Failed or incomplete placental spiral artery remodeling (SAR) can contribute to life-threatening maternal hypertension and fetal growth restrictions in the later stages of pregnancy. A critical step in SAR is the degradation of extracellular matrix and the loss of vascular smooth muscle coverage, and it?s well established that uterine natural killer (uNK) cells are essential for the completion of this step. Unfortunately, the molecular mechanisms underlying this process, and the role of the maternal endothelium in particular, are not well understood. One factor secreted by uNK cells is VEGFC, a key lymphangiogenic factor, and a peculiarity given the lack of lymphatic development in the placental decidua where spiral arteries are located. Historically, the VEGFC receptor, VEGFR3, has been considered to be restricted to lymphatic vessels in adults. However, the recent discovery of hybrid vessels that express a combination of blood and lymphatic markers prompted me to investigate if VEGFR3 is expressed in spiral arteries. My preliminary work has identified the expression of VEGFR3 on the spiral artery endothelium, and that the timing of its initial expression coincides with SAR. What remains unclear is what role endothelial VEGFR3 expression may have, and whether or not other lymphatic markers are also being expressed in spiral arteries. Therefore, Aim 1 will define the spatial and temporal relationship of lymphatic expression to actions essential for successful SAR in both mouse and human placentas. In Aim 1, I propose to use immunohistochemistry and immunoblotting to examine the expression of a key subset of lymphatic markers in spiral arteries while using advanced high throughput expression analysis to identify the broader shift in blood and lymphatic expression that occurs in the spiral artery endothelium during SAR. Aim 2 will define the mechanism of, and subsequent downstream response to, VEGFR3 activation in spiral arteries, and whether that activation contributes to the regulation of smooth muscle coverage on spiral arteries. In Aim 2, I will use loss-of-function mouse models and in vitro assays to characterize the contribution of uNK cells to VEGFR3 activation. Specifically, I will use IL2R? null mice that lack uNK cells for the in vivo examination and uNK-conditioned media treatments on VEGFR3-expressin endothelial cells for in vitro analysis. I will then determine if VEGFR3 regulates spiral artery smooth muscle coverage by examining SAR in mice expressing a dominant negative kinase dead version of VEGFR and by triggering VEGFR3 activation in endothelial cells co- cultured with smooth muscle cells in vitro. Results from these experiments will address the broader role of lymphatic expression in spiral arteries while revealing a novel mechanism by which uNK cells promote SAR. Ultimately, this work will identify new targets for the treatment for the pathological origins of pre-eclampsia.